Controls for proximity detection assays

ABSTRACT

The present invention provides a method for detecting a plurality of analytes in a sample, comprising performing a multiplex proximity-based detection assay. The assay utilises pairs of proximity probes with shared hybridisation sites (i.e. hybridisation sites which are shared between different proximity probe pairs). Also provided is a product comprising a plurality of proximity probe pairs with shared hybridisation sites, which may be used in the method disclosed herein.

FIELD

The present invention provides a method for detecting a plurality ofanalytes in a sample, comprising performing a multiplex proximity-baseddetection assay. The assay utilises pairs of proximity probes withshared hybridisation sites (i.e. hybridisation sites which are sharedbetween different proximity probe pairs). Also provided is a productcomprising a plurality of proximity probe pairs with sharedhybridisation sites, which may be used in the method disclosed herein.

BACKGROUND

Modern proteomics methods require the ability to detect a large numberof different proteins (or protein complexes) in a small sample volume.To achieve this, multiplex analysis must be performed. Common methods bywhich multiplex detection of proteins in a sample may be achievedinclude proximity extension assays (PEA) and proximity ligation assays(PLA). PEA and PLA are described in WO 01/61037; PEA is furtherdescribed in WO 03/044231, WO 2004/094456, WO 2005/123963, WO2006/137932 and WO 2013/113699.

PEA and PLA are proximity assays, which rely on the principle of“proximity probing”. In these methods an analyte is detected by thebinding of multiple (i.e. two or more, generally two or three) probes,which when brought into proximity by binding to the analyte (hence“proximity probes”) allow a signal to be generated. Typically, at leastone of the proximity probes comprises a nucleic acid domain (or moiety)linked to the analyte-binding domain (or moiety) of the probe, andgeneration of the signal involves an interaction between the nucleicacid moieties and/or a further functional moiety which is carried by theother probe(s). Thus signal generation is dependent on an interactionbetween the probes (more particularly between the nucleic acid or otherfunctional moieties/domains carried by them) and hence only occurs whenthe necessary probes have bound to the analyte, thereby lending improvedspecificity to the detection system.

In PEA, nucleic acid moieties linked to the analyte-binding domains of aprobe pair hybridise to one another when the probes are in closeproximity (i.e. when bound to a target), and are then extended using anucleic acid polymerase. The nucleic acid moieties of the probes withina probe pair comprise complementary “hybridisation sites”, whichhybridise to one another. The extension product forms a reporter nucleicacid, detection of which demonstrates the presence in a sample ofinterest of a particular analyte (the analyte bound by the relevantprobe pair).

In PLA, nucleic acid moieties linked to the analyte-binding domains of aprobe pair come into proximity when the probes of the probe pair bindtheir target, and may be ligated together, or alternatively they maytogether template the ligation of separately added oligonucleotideswhich are able to hybridise to the nucleic acid domains when they are inproximity. In PLA methods at least one “splint” oligonucleotide isprovided, which bridges the proximity probe nucleic acid moieties. Thesplint oligonucleotide comprises sequences which are complementary to“hybridisation sites” on the probe nucleic acid domains. Binding of theprobe nucleic acid moieties to the splint oligonucleotide enables theligation together of the two probe nucleic acid moieties. Alternatively,as mentioned above, a second splint molecule may be added and ligated tothe first splint. The ligation product is then amplified, acting as areporter nucleic acid.

Multiplex analyte detection using PEA or PLA may be achieved byincluding a unique identifier (ID) sequence, such as a barcode sequenceor a primer or probe binding site, in the nucleic acid moiety of eachprobe. A reporter nucleic acid molecule corresponding to a particularanalyte may be identified by the ID sequences it contains.

Some “background” (i.e. false positive) signal is inevitable inproximity assays. Background signal may occur as a result of randominteractions with or between unbound proximity probes in the reactionsolution. Currently, the level of background signal in a proximityreaction is determined by the use of a separate negative control. Forthe negative control a proximity assay is performed using just buffer(i.e. no sample), such that all signal is background. Comparison ofexperimental assays to the negative control allows the true positivesignal to be determined.

The present invention provides a method of performing a multiplexproximity assay with an improved background control. In this method,different proximity probe pairs share hybridisation sites. Thisencourages the formation of “background” signal between all unboundprobes sharing the same hybridisation sites. All signal from generatedreporter nucleic acids is read together (both true and false positive).True positive signal can be distinguished from false positive signalbased on whether the resulting reporter nucleic acid comprises pairedbarcode sequences (i.e. barcode sequences each corresponding to the sameanalyte, indicating a true positive signal) or unpaired barcodesequences (i.e. barcode sequences corresponding to different analytes,indicating a false positive signal). The level of false positive signalgenerated in the reaction indicates the level of background, meaningthat a separate negative control reaction to determine background levelno longer needs to be performed, simplifying the overall assay.

The use of shared hybridisation sites to determine background alsomitigates against differences in the performance between differenthybridisation sites. Different pairs of hybridisation sites may interactmore or less strongly than others, resulting in different levels ofbackground being produced from each pair of hybridisation sites. Theshared hybridisation sites allow the level of background generated fromeach hybridisation site pair to be individually determined, resulting ina more accurate determination of the level of background to becalculated. The present invention thus provides a more straightforwardand accurate means of controlling for false positive results inproximity assays.

SUMMARY OF INVENTION

To this end, in a first aspect the present invention provides a methodfor detecting a plurality of analytes in a sample, the method comprisingperforming a multiplex proximity-based detection assay, the assaycomprising:

(i) contacting the sample with a plurality of pairs of proximity probes,wherein each proximity probe pair comprises a first proximity probe anda second proximity probe, and each proximity probe comprises:

-   -   (a) an analyte-binding domain specific for an analyte; and    -   (b) a nucleic acid domain,

wherein both probes within each pair comprise analyte-binding domainsspecific for the same analyte, and can simultaneously bind to theanalyte; and each probe pair is specific for a different analyte;

wherein the nucleic acid domain of each proximity probe comprises an IDsequence and at least a first hybridisation sequence, wherein the IDsequence of each proximity probe is different; and wherein:

in each proximity probe pair, the first proximity probe and the secondproximity probe comprise paired hybridisation sequences, such that uponbinding of the first and second proximity probe to their analyte, therespective paired hybridisation sequences of the first and secondproximity probes hybridise to each other or to a common splintoligonucleotide which comprises hybridisation sequences complementary toeach of the paired hybridisation sequences of the first and secondproximity probes;

and wherein at least one pair of hybridisation sequences is shared by atleast two pairs of proximity probes;

(ii) allowing the nucleic acid domains of the proximity probes tohybridise to one another or to the splint oligonucleotide, to form acontinuous or non-continuous duplex comprising the hybridisationsequence of a first proximity probe and a hybridisation sequence of asecond proximity probe, wherein said duplex comprises at least one free3′ end;

(iii) subjecting the duplex to an extension and/or ligation reaction togenerate an extension and/or ligation product which comprises the IDsequence of the first proximity probe and the ID sequence of the secondproximity probe

(iv) amplifying the extension product or ligation product;

(v) detecting the extension product or ligation product, whereindetection of the extension product or ligation product comprisesidentification of the ID sequences therein, and determining the relativeamounts of each extension product or ligation product; and

(vi) determining which analytes are present in the sample, wherein:

-   -   (a) extension products and/or ligation products which comprise a        first ID sequence from a first proximity probe belonging to a        first proximity probe pair and a second ID sequence from a        second proximity probe belonging to a second proximity probe        pair are deemed background; and    -   (b) an extension product or ligation product which comprises a        first ID sequence and a second ID sequence from a proximity        probe pair, and which is present in an amount higher than the        background, indicates that the analyte specifically bound by the        proximity probe pair is present in the sample.

In a second aspect the present invention provides a product comprising:

(i) a plurality of proximity probe pairs, wherein each proximity probepair comprises a first proximity probe and a second proximity probe, andeach proximity probe comprises:

-   -   (a) a protein-binding domain specific for a protein; and    -   (b) a nucleic acid domain,

wherein both probes within each pair comprise protein-binding domainsspecific for the same protein, and can simultaneously bind to theprotein; and each probe pair is specific for a different protein;

wherein the nucleic acid domain of each proximity probe comprises an IDsequence and at least a first hybridisation sequence, wherein the IDsequence of each proximity probe is different; and wherein in eachproximity probe pair, the first proximity probe and the second proximityprobe comprise paired hybridisation sequences; and, optionally

(ii) a plurality of splint oligonucleotides, each splint oligonucleotidecomprising hybridisation sequences complementary to each of the pairedhybridisation sequences of a proximity probe pair;

wherein the hybridisation sequences of each proximity probe pair areconfigured such that upon binding of the first and second proximityprobe to their protein, the respective paired hybridisation sequences ofthe first and second proximity probes hybridise to each other or to asplint oligonucleotide;

and wherein at least one pair of hybridisation sequences is shared by atleast two pairs of proximity probes.

DETAILED DESCRIPTION

As detailed above, the first aspect of the invention provides a methodfor detecting a plurality of analytes in a sample. The term “analyte” asused herein means any substance (e.g. molecule) or entity it is desiredto detect by the method of the invention. The analyte is thus the“target” of the assay method of the invention, i.e. the substancedetected or screened for using the method of the invention.

The analyte may accordingly be any biomolecule or chemical compound itis desired to detect, for example a peptide or protein, or a nucleicacid molecule or a small molecule, including organic and inorganicmolecules. The analyte may be a cell or a microorganism, including avirus, or a fragment or product thereof. It will be seen therefore thatthe analyte can be any substance or entity for which a specific bindingpartner (e.g. an affinity binding partner) can be developed. All that isrequired is that the analyte is capable of simultaneously binding atleast two binding partners (more particularly, the analyte-bindingdomains of at least two proximity probes).

Proximity probe-based assays have found particular utility in thedetection of proteins or polypeptides. Analytes of particular interestthus include proteinaceous molecules such as peptides, polypeptides,proteins or prions or any molecule which includes a protein orpolypeptide component, etc., or fragments thereof. In a particularlypreferred embodiment of the invention, the analyte is a wholly orpartially proteinaceous molecule, most particularly a protein. That isto say, it is preferred that the analyte is or comprises a protein.

The analyte may be a single molecule or a complex that contains two ormore molecular subunits, which may or may not be covalently bound to oneanother, and which may be the same or different. Thus in addition tocells or microorganisms, such a complex analyte may also be a proteincomplex, or a biomolecular complex comprising a protein and one or moreother types of biomolecule. Such a complex may thus be a homo- orhetero-multimer. Aggregates of molecules, e.g. proteins, may also betarget analytes, for example aggregates of the same protein or differentproteins. The analyte may also be a complex between proteins or peptidesand nucleic acid molecules such as DNA or RNA. Of particular interestmay be the interactions between proteins and nucleic acids, e.g.regulatory factors, such as transcription factors, and DNA or RNA. Thusin a particular embodiment the analyte is a protein-nucleic acid complex(e.g. a protein-DNA complex or a protein-RNA complex). In anotherembodiment, the analyte is a non-nucleic acid analyte, by which is meantan analyte which does not comprise a nucleic acid molecule. Non-nucleicacid analytes include proteins and protein complexes, as mentionedabove, small molecules and lipids.

The method of the invention is directed to detecting a plurality ofanalytes in a sample. The plurality of analytes may be of the same type(e.g. all the analytes may be proteins, or protein complexes), or ofdifferent types (e.g. some analytes may be proteins, others proteincomplexes, others lipids, others protein-DNA or protein-RNA complexes,etc., or any combination of such types of analytes).

The term “a plurality of” as used in the present disclosure means morethan one (that is to say, two or more), in line with its standarddefinition. The terms “a plurality of” and “multiple” areinterchangeable. Thus the method of the invention is used to detect atleast two analytes in a sample. However, it is preferred thatconsiderably more analytes than two are detected according to thepresent method. Preferably at least 10, 20, 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or moreanalytes are detected according to the present method.

The term “detecting” or “detected” is used broadly herein to include anymeans of determining the presence or absence of an analyte (i.e.determining whether a target analyte is present in a sample of interestor not). Accordingly, if a method of the invention is performed and anattempt is made to detect a particular analyte of interest in a sample,but the analyte is not detected because it is not present in the sample,the step of “detecting the analyte” has still been performed, becauseits presence or absence from the sample has been assessed. The step of“detecting” an analyte is not dependent on that detection provingsuccessful, i.e. on the analyte actually being detected.

Detecting an analyte may further include any form of measurement of theconcentration or abundance of the analyte in the sample. Either theabsolute concentration of a target analyte may be determined, or arelative concentration of the analyte, for which purpose theconcentration of the target analyte may be compared to the concentrationof another target analyte (or other target analytes) in the sample or inother samples. Thus “detecting” may include determining, measuring,assessing or assaying the presence or absence or amount of an analyte inany way. Quantitative and qualitative determinations, measurements orassessments are included, including semi-quantitative determinations.Such determinations, measurements or assessments may be relative, forexample when two or more different analytes in a sample are beingdetected, or absolute. As such, the term “quantifying” when used in thecontext of quantifying a target analyte in a sample can refer toabsolute or to relative quantification. Absolute quantification may beaccomplished by inclusion of known concentration(s) of one or morecontrol analytes and/or referencing the detected level of the targetanalyte with known control analytes (e.g. through generation of astandard curve). Alternatively, relative quantification can beaccomplished by comparison of detected levels or amounts between two ormore different target analytes to provide a relative quantification ofeach of the two or more different analytes, i.e. relative to each other.Similarly, the relative levels of a particular analyte in two differentsamples may be quantified. Methods by which quantification can beachieved in the method of the invention are discussed further below.

The method of the invention is for detecting multiple analytes in asample. Any sample of interest may be assayed according to theinvention. That is to say any sample which contains or may containanalytes of interest, and which a person wishes to analyse to determinewhether or not it contains analytes of interest, and/or to determine theconcentrations of analytes of interest therein.

Any biological or clinical sample may thus be analysed according to thepresent invention, e.g. any cell or tissue sample of or from anorganism, or any body fluid or preparation derived therefrom, as well assamples such as cell cultures, cell preparations, cell lysates etc.Environmental samples, e.g. soil and water samples, or food samples mayalso be analysed according to the invention. The samples may be freshlyprepared or they may be prior-treated in any convenient way e.g. forstorage.

Representative samples thus include any material which may contain abiomolecule, or any other desired or target analyte, including forexample foods and allied products, clinical and environmental samples.The sample may be a biological sample, which may contain any viral orcellular material, including prokaryotic or eukaryotic cells, viruses,bacteriophages, mycoplasmas, protoplasts and organelles. Such biologicalmaterial may thus comprise any type of mammalian and/or non-mammaliananimal cell, plant cells, algae including blue-green algae, fungi,bacteria, protozoa etc.

It is preferred that the sample is a clinical sample, for instance wholeblood and blood-derived products such as plasma, serum, buffy coat andblood cells, urine, faeces, cerebrospinal fluid or any other body fluid(e.g. respiratory secretions, saliva, milk etc.), tissues and biopsies.It is particularly preferred that the sample is a plasma or serumsample. Thus the method of the invention may be used in the detection ofbiomarkers, for instance, or to assay a sample for pathogen-derivedanalytes. The sample may in particular be derived from a human, thoughthe method of the invention may equally be applied to samples derivedfrom non-human animals (i.e. veterinary samples). The sample may bepre-treated in any convenient or desired way to prepare it for use inthe method of the invention, for example by cell lysis or removal, etc.

The method of the invention comprises performing a multiplexproximity-based detection assay. As used herein, the term “multiplex” isused to refer to an assay in which multiple (i.e. at least two)different analytes are assayed at the same time in the same reactionmixture. Preferably, however, considerably more than two analytes areassayed in a multiplex reaction according to the invention. Forinstance, a multiplex reaction may assay at least 5, 10, 15, 20, 25, 30,40, 50, 60 analytes or more. Certain multiplex reactions may assay morethan this number of analytes, e.g. at least 70, 80, 90, 100, 110, 120,130, 140 or 150 analytes or more.

A “proximity-based detection assay” is any assay which utilisesproximity probes to detect an analyte in a sample. Generally speaking aproximity probe is a probe which interacts with at least one other,cognate, proximity probe, to produce a signal which may be detected inorder to detect an analyte. Proximity probes are well known in the art.A proximity probe as used according to the present disclosure andinvention and as defined in the claims herein is an entity comprising ananalyte-binding domain specific for an analyte, and a nucleic aciddomain. By “specific for an analyte” is meant that the analyte-bindingdomain specifically recognises and binds a particular target analyte,i.e. it binds its target analyte with higher affinity than it binds toother analytes or moieties. The analyte-binding domain is preferably anantibody, in particular a monoclonal antibody. Antibody fragments orderivatives of antibodies comprising the antigen-binding domain are alsosuitable for use as the analyte binding domain. Examples of suchantibody fragments or derivatives include Fab, Fab′, F(ab′)₂ and scFvmolecules.

A Fab fragment consists of the antigen-binding domain of an antibody. Anindividual antibody may be seen to contain two Fab fragments, eachconsisting of a light chain and its conjoined N-terminal section of theheavy chain. Thus a Fab fragment contains an entire light chain and theV_(H) and C_(H)1 domains of the heavy chain to which it is bound. Fabfragments may be obtained by digesting an antibody with papain.

F(ab′)₂ fragments consist of the two Fab fragments of an antibody, plusthe hinge regions of the heavy domains, including the disulphide bondslinking the two heavy chains together. In other words, a F(ab′)₂fragment can be seen as two covalently joined Fab fragments. F(ab′)₂fragments may be obtained by digesting an antibody with pepsin.Reduction of F(ab′)₂ fragments yields two Fab′ fragments, which can beseen as Fab fragments containing an additional sulfhydryl group whichcan be useful for conjugation of the fragment to other molecules. ScFvmolecules are synthetic constructs produced by fusing together thevariable domains of the light and heavy chains of an antibody.Typically, this fusion is achieved recombinantly, by engineering theantibody gene to produce a fusion protein which comprises both the heavyand light chain variable domains.

The nucleic acid domain of a proximity probe may be a DNA domain or anRNA domain. Preferably it is a DNA domain. The nucleic acid domains ofthe proximity probes in each pair typically are designed to hybridise toone another, or to one or more common oligonucleotide molecules (towhich the nucleic acid domains of both proximity probes of a pair mayhybridise). Accordingly, the nucleic acid domains must be at leastpartially single-stranded. In certain embodiments the nucleic aciddomains of the proximity probes are wholly single-stranded. In otherembodiments, the nucleic acid domains of the proximity probes arepartially single-stranded, comprising both a single-stranded part and adouble-stranded part.

Proximity probes are typically provided in pairs, each pair beingspecific for a target analyte. As noted above, a target analyte may be asingle entity, in particular an individual protein. In this embodiment,both probes in the proximity pair bind the target analyte (e.g.protein), but at different epitopes. The epitopes are non-overlapping,so that the binding of one probe in the pair to its epitope does notinterfere with or block binding of the other probe in the pair to itsepitope. Alternatively, as noted above the target analyte may be acomplex, e.g. a protein complex, in which case one probe in the pairbinds one member of the complex and the other probe in the pair bindsthe other member of the complex. The probes bind the proteins within thecomplex at sites different to the interaction sites of the proteins(i.e. the sites in the proteins through which they interact with eachother).

As noted above, proximity probes are provided in pairs, each specificfor a target analyte. By this is meant that within each proximity probepair, both probes comprise analyte-binding domains specific for the sameanalyte. Since the detection assay used is a multiplex assay, multipledifferent probe pairs are used in each detection assay, each probe pairbeing specific for a different analyte. That is to say, theanalyte-binding domains of each different probe pair are specific for adifferent target analyte. Any detection method utilising proximityprobes may be used according to the present invention. As detailedabove, particularly suitable proximity-based detection assays areproximity extension assays (PEA) and proximity ligation assays (PLA).

The method of the invention comprises a first step of contacting thesample with a plurality of (i.e. multiple) pairs of proximity probes.Each proximity probe pair comprises a first proximity probe and a secondproximity probe, and each proximity probe comprises: (a) ananalyte-binding domain specific for an analyte; and (b) a nucleic aciddomain. In each proximity probe pair, both probes compriseanalyte-binding domains specific for the same analyte, and each probepair is specific for a different analyte (i.e. each probe pair comprisesanalyte-binding domains which are specific for a different analyte).

The nucleic acid domain of each proximity probe comprises anidentification (ID) sequence. Each proximity probe comprises a unique IDsequence (i.e. a different ID sequence is present in each proximityprobe). Notably, this does not mean that each individual probe moleculecomprises a unique ID sequence. Rather, each probe species comprises aunique ID sequence. By “probe species” is meant a probe comprising aparticular analyte-binding domain, and thus in other words, all probemolecules comprising the same analyte-binding domain comprise the sameunique ID sequence. Every different probe species comprises a differentID sequence. As further discussed below, the ID sequences allowidentification of reporter nucleic acids generated in the method of theinvention.

The nucleic acid domain of each proximity probe also comprises at leastone (or at least a first) hybridisation sequence. The firsthybridisation sequences (which may be the only hybridisation sequencesin the proximity probes, depending on the structures of the probes used)are paired within each proximity probe pair. By “paired hybridisationsequences” is meant that the two hybridisation sequences within the pairare capable of directly or indirectly interacting with each other, suchthat when the method of the invention is performed and a pair ofproximity probes bind to their target analyte, the nucleic acid domainsof the two probes become directly or indirectly linked to one another.

In a particular and preferred embodiment, paired hybridisation sequencesare complementary to one another, such that they hybridise to oneanother. In this embodiment, the hybridisation sequence of the firstproximity probe in a pair is the reverse complement of the hybridisationsequence of the second proximity probe in the pair.

In an alternative embodiment, the paired hybridisation sequences do nothybridise directly to one another, but instead both hybridise to aseparate, bridging oligonucleotide, referred to herein as a splintoligonucleotide. The separate oligonucleotide may be regarded as a thirdoligonucleotide in the assay method. However, one or more splintoligonucleotides may be used, and thus there may be third or furtheroligonucleotides to which the paired hybridisation sequences mayhybridise. In other words, the paired hybridisation sequences are ableto hybridise to a common oligonucleotide. This may be a templateoligonucleotide which is capable of templating the ligation and/orextension of the nucleic acid domains, or it may be that the extensionand/or ligation of the third and optionally further oligonucleotides istemplated by the nucleic acid domains.

In one such embodiment, along with the pair of proximity probes thesplint oligonucleotide may form a third member of each proximity assayset. The splint oligonucleotide comprises two hybridisation sequences:one complementary to the hybridisation sequence of the first probe inthe probe pair, and the other complementary to the hybridisationsequence of the second probe in the probe pair. The splintoligonucleotide is thus capable of hybridising to both of the pairedhybridisation sequences of the proximity probes in its proximity assayset. Notably, the splint oligonucleotide is capable of hybridising toboth of the paired hybridisation sequences of the proximity probes inits proximity assay set at the same time. Accordingly, when a pair ofproximity probes bind their analyte and come into proximity, the nucleicacid domains of the probes both hybridise to the splint oligonucleotide,thus forming a complex comprising the two probe nucleic acid domains andthe splint oligonucleotide.

In the present method, at least one pair of hybridisation sequences isshared by at least two pairs of proximity probes. In other words, atleast two pairs of proximity probes (which bind to different analytes)have the same hybridisation sequences. Probes from pairs which share apair of hybridisation sequences are capable of hybridising to eachother, or forming a complex together. Hybridisation is most likely tooccur between the nucleic acid domains of a pair of proximity probeswhen they are both bound to their respective analyte, since binding ofthe probes to the analyte brings the nucleic acid domains into closeproximity. However, some interactions will inevitably form betweenpaired hybridisation sequences of the nucleic acid domains of unboundproximity probes in solution (i.e. the nucleic acid domains of proximityprobes which are not bound to their analyte), or when only one proximityprobe has bound to its target analyte it may interact with another probein solution. Notably, in solution the nucleic acid domain of an unboundproximity probe is equally likely to hybridise to (or form a complexwith) the nucleic acid domain of any proximity probe which has a pairedhybridisation sequence, regardless of whether the proximity probe bindsthe same analyte or a different analyte. Reporter nucleic acidsgenerated as a result of such non-specific hybridisation (i.e. as aresult of hybridisation between unbound proximity probes in solution)form background, as described further below.

It is preferred that a significant proportion of probe pairs share theirhybridisation sequences with at least one other proximity probe pair. Inparticular embodiments, at least 25%, 50% or 75% of proximity probepairs share their hybridisation sequences with another proximity probepair (i.e. with at least one other proximity probe pair). In aparticular embodiment, all proximity probe pairs share theirhybridisation sequences with at least one other proximity probe pair.However, as is apparent from the above, in another embodiment at leastone pair of hybridisation sequences is unique to a single pair ofproximity probes. That is to say, at least one pair of proximity probesdoes not share its hybridisation sequences with any other proximityprobe pair. In particular embodiments, up to 75%, 50% or 25% of pairs ofproximity probes do not share their hybridisation sequences with anyother proximity probe pair.

In an embodiment of the invention, a single pair of hybridisationsequences is shared across all probe pairs which have sharedhybridisation sequences. That is to say, all probe pairs which sharetheir hybridisation sequences with another probe pair have the same pairof hybridisation sequences. In this embodiment, potentially all probepairs used in the multiplex assay may have the same pair ofhybridisation sequences.

However, if too many probe pairs share the same pair of hybridisationsequences, this can allow too large a number of background interactionsto take place, hiding the true positive signals. Accordingly, it may bepreferred that each pair of hybridisation sequences is shared by a morelimited number of probe pairs. In particular embodiments, no more than20, 15, 10 or 5 proximity probe pairs share the same pair ofhybridisation sequences. Thus it is preferred that the multiplex assayof the invention uses multiple sets of proximity probe pairs, each ofwhich share a particular pair of hybridisation sequences. Thus allproximity probe pairs in a particular proximity probe pair set share thesame pair of hybridisation sequences, but a different pair ofhybridisation sequences is used by each different proximity probe pairset. This enables non-specific hybridisation between all probe pairswithin each probe pair set, but prevents non-specific hybridisationbetween probe pairs in different probe pair sets. In general, each probepair set comprises in the range 2 to 5 probe pairs, though larger setsmay be used if preferred.

The number of probe pair sets used in any given multiplex assay isdependent on the total number of probe pairs used in the assay, i.e. thenumber of different analytes detected in the assay. Inevitably, thegreater the number of probe pairs used in the assay, the greater thenumber of probe pair sets.

The first step of the method of the invention comprises contacting thesample with the plurality of pairs of proximity probes discussed above.The proximity probes of a pair may be added to the sample pre-mixed aspairs, or as individual proximity probes. That is, the sample may becontacted with the proximity probes of a pair separately or together atthe same time, either by contacting the probes simultaneously or in thesame reaction mixture. If the proximity probes are configured such thatboth probes in a probe pair hybridise to a common splint oligonucleotide(rather than to each other), the various splint oligonucleotides may beincluded with the proximity probe pairs, or with one of the proximityprobes of a pair, or may be added separately at the same time, or afterthe proximity probes. “Contacting the sample” means that the sample andthe proximity probe pairs are mixed. The proximity probe pairs may beadded to the sample, or conversely the sample may be added to theproximity probe pairs. The sample may be diluted, prior to contactingwith the proximity probe pairs. If dilution of the sample is required,this may be performed using an appropriate diluent, for instance abuffer. Suitable buffers for use as diluent include PBS(phosphate-buffered saline), TBS (Tris-buffered saline), HBS(HEPES-buffered saline), etc. The buffer (or other diluent) used must bemade up in a purified solvent (e.g. water) such that it does not containcontaminant analytes. The diluent should thus be sterile, and if wateris used as diluent or the base of the diluent, the water used ispreferably ultrapure (e.g. Milli-Q water).

Following contacting of the sample with the proximity probe pairs, thenucleic acid domains of the proximity probes are allowed to hybridise toone another, or to the splint oligonucleotide, as appropriate.Hybridisation of the nucleic acid domains to one another or to thesplint oligonucleotide results in the formation of a continuous ornon-continuous duplex. A “duplex” as referred to herein is a section ofdouble-stranded nucleic acid. The duplex comprises the hybridisationsequence of a first proximity probe and the hybridisation sequence of asecond proximity probe. If the hybridisation sequences hybridise to acommon splint oligonucleotide, rather than to each other, the duplexalso comprises the common splint oligonucleotide.

In this step, hybridisation of the nucleic acid domains to one anotherresults in the formation of a continuous duplex, that is to say a singleduplex comprising the entirety of the hybridisation sequences of bothnucleic acid domains. Hybridisation of the probe nucleic acid domains toa common splint oligonucleotide results in the formation of adiscontinuous duplex, comprising a first part formed between the splintoligonucleotide and the hybridisation sequence of the first probe, asecond part formed between the splint oligonucleotide and thehybridisation sequence of the second probe, and a gap located betweenthe first and second parts of the duplex (i.e. between the hybridisationsequences of the two probes). The discontinuous duplex may alternativelybe seen as two separate duplexes (i.e. the first and second parts of thediscontinuous duplex may alternatively be seen as separate first andsecond duplexes). Seen in this manner, hybridisation of the probenucleic acid domains to a common splint oligonucleotide results in theformation of two linked duplexes. The duplexes are linked in that theyare joined by the common splint oligonucleotide.

The duplex generated by hybridisation of the nucleic acid domains to oneanother, or to the common splint oligonucleotide, comprises a free 3′end (or at least one free 3′ end—the duplex may contain multiple free 3′ends. In certain embodiments the duplex comprises two free 3′ ends). Afree 3′ end is a 3′ end of a nucleic acid strand in a duplex which iscapable of being extended by a polymerase.

In this step, hybridisation generally and most frequently occurs betweenthe nucleic acid domains of proximity probes in a proximity probe pair,which are bound to a target analyte. However, as described above,background hybridisation also occurs with or between the nucleic aciddomains of unbound, unpaired probes in solution. Such backgroundhybridisation occurs between the nucleic acid domains of probes fromprobe pairs which share common hybridisation sequences.

Following hybridisation of the nucleic acid domains to form the duplex,the duplex is subjected to an extension and/or ligation reaction togenerate an extension and/or ligation product which comprises the IDsequence of the first proximity probe and the ID sequence of the secondproximity probe. The nature of the reactions performed is dependent onwhether the proximity assay performed is a PLA or PEA. In the context ofa PEA, only an extension reaction is performed, thus generating anextension product. A number of PEA variants are discussed below. In thecontext of a PLA, a ligation reaction is performed, but an extensionreaction may also be performed. Variants of PLA are also discussedbelow. Preferably, the extension and/or ligation product is a linearextension and/or ligation product (i.e. it is not a circular product).

Once the extension product or ligation product has been generated, it isamplified. Amplification may be performed using any known nucleic acidamplification technique. Preferably, amplification is performed by PCR,though any other method of nucleic acid amplification may be utilised,e.g. loop-mediated isothermal amplification (LAMP).

In a preferred embodiment, all reporter nucleic acids generated in themultiplex assay (i.e. extension and/or ligation products) comprisecommon primer binding sites. That is to say, all reporter nucleic acidsgenerated comprise the same pair of primer binding sites. This isadvantageous as it allows all reporter nucleic acids generated to beamplified in a single amplification reaction (e.g. PCR) using a singleprimer pair.

Once amplified, the reporter nucleic acid is detected. Detection of thereporter nucleic acid is achieved by detecting the ID sequences therein.By detecting the ID sequences within the extension or ligation product,it can be determined which probes hybridised to each other to generatethe product. The relative amounts of each extension or ligation productare also determined in this step. Any suitable detection method known inthe art may be used.

The ID sequence may be any sequence by which a proximity probe may bedistinguished or identified. It is thus a tag sequence by which aparticular proximity probe may be detected. The ID sequence may bedirectly directed, or it may provide a binding site for a further entityby which it may be detected, e.g. for a specific primer, or a detectionprobe.

In a preferred embodiment of the invention, the ID sequences are barcodesequences. A barcode sequence is a particular nucleotide sequence whichis defined to correspond to a particular analyte. If each probe carriesa barcode sequence, each reporter nucleic acid will contain two barcodesequences: one from each of the two probes which combined to yield theproduct. When the two barcode sequences are detected, the two probeswhich combined to yield the reporter nucleic acid can thus beidentified. If the two barcode sequences are from a proximity probe pair(i.e. from a pair of probes which bind the same target analyte), thereporter nucleic acid may indicate the presence of the target analyte inthe sample, or may be background. If the two barcode sequences are fromunpaired proximity probes (i.e. the two barcode sequences are indicativeof different analytes), the reporter nucleic acid is deemed background.

The barcode sequences are located within the nucleic acid domains of theprobes. The barcode sequence is not located within the firsthybridisation sequence: as detailed above, each proximity probecomprises a different barcode sequence, whereas the hybridisationsequences are shared between multiple different probes. Barcodesequences are also not located in the common primer binding sites—asnoted above, each probe comprises a unique barcode sequence, whereas itis preferred that all probes comprise common primer binding sites.

Barcode sequences can be detected in a number of ways. Firstly, specificbarcode sequences may be detected by sequencing of all the reporternucleic acid molecules generated during the multiplex detection assay.By sequencing all the reporter nucleic acid molecules generated, all thedifferent reporter nucleic acid molecules generated may be identified bytheir barcode sequences. Nucleic acid sequencing is the preferred methodof reporter nucleic acid detection/analysis.

Other suitable methods for detecting barcodes in the reporter nucleicacid molecules include PCR-based methods. For instance, quantitative PCRutilising “TaqMan” probes may be performed. In this instance, thereporter nucleic acid molecules (or at least a section of each reporternucleic acid molecule comprising the barcode sequences) is amplified,and a probe complementary to each barcode sequence is provided, witheach different probe being conjugated to a different, distinguishablefluorophore. The presence or absence of each barcode can then bedetermined based on whether the particular barcode is amplified.However, it is apparent that PCR-based methods such as described aboveare only suitable for analysis of relatively small numbers of differentsequences at the same time, although combinatorial methods using probesfor decoding barcode sequences are known and may be used to extendmultiplexing capacity to a degree. Nucleic acid sequencing does not haveany real limit on the number of sequences which can be identified in anyone go, enabling higher levels of multiplex reaction than detectionusing PCR, hence sequencing is the preferred method for reporter nucleicacid molecule detection.

Preferably, a form of high throughput DNA sequencing is used to detectbarcodes in the reporter nucleic acid molecules. Sequencing by synthesisis the preferred DNA sequencing method. Examples of sequencing bysynthesis techniques include pyrosequencing, reversible dye terminatorsequencing and ion torrent sequencing, any of which may be utilised inthe present method. Preferably the reporter nucleic acids are sequencedusing massively parallel DNA sequencing. Massively parallel DNAsequencing may in particular be applied to sequencing by synthesis (e.g.reversible dye terminator sequencing, pyrosequencing or ion torrentsequencing, as mentioned above). Massively parallel DNA sequencing usingthe reversible dye terminator method is a preferred sequencing method.Massively parallel DNA sequencing using the reversible dye terminatormethod may be performed, for instance, using an Illumina® NovaSeq™system.

As is known in the art, massively parallel DNA sequencing is a techniquein which multiple (e.g. thousands or millions or more) DNA strands aresequenced in parallel, i.e. at the same time. Massively parallel DNAsequencing requires target DNA molecules to be immobilised to a solidsurface, e.g. to the surface of a flow cell or to a bead. Eachimmobilised DNA molecule is then individually sequenced. Generally,massively parallel DNA sequencing employing reversible dye terminatorsequencing utilises a flow cell as the immobilisation surface, andmassively parallel DNA sequencing employing pyrosequencing or iontorrent sequencing utilises a bead as the immobilisation surface.

As is known to the skilled person, immobilisation of DNA molecules to asurface in the context of massively parallel sequencing is generallyachieved by the attachment of one or more sequencing adapters to theends of the molecules, which are capable of attaching the DNA moleculesto a target surface. The method of the invention may thus include theaddition of one or more adapters for sequencing (sequencing adapters) tothe reporter nucleic acid molecules, as described in more detail below.

In alternative embodiment, the ID sequences are not barcode sequences.Rather, the ID sequences may allow identification of the probes by othermeans. Depending on the nature of the ID sequence, any suitable methodmay be used to identify the ID sequence. For instance, the ID sequencesmay be restriction sites (i.e. a nucleotide sequence recognised by arestriction enzyme). In this embodiment, the nucleic acid domain of eachproximity probe comprises a different restriction site (such that it isrecognised and cleaved by a different restriction enzyme). Differentcombinations of restriction enzymes may thus be applied to the reporternucleic acids generated from the multiplex assay, to determine whatcombinations of probes have interacted. If a reporter nucleic acid iscleaved by both of a pair restriction enzymes applied, this demonstratesthat the two probes which comprise the respective restriction sites forthe enzymes have interacted to yield a reporter nucleic acid.

In another embodiment, the ID sequences are primer binding sites. Inthis embodiment, the nucleic acid domain of each proximity probecomprises a unique primer binding site. Amplification of the reporternucleic acid molecules with different combinations of primers is thenperformed to determine what combinations of probes have interacted. Ifan amplification reaction with a particular primer pair yields anamplification product, this demonstrates that the two probes whichcomprise the respective primer binding sites have interacted to generatea reporter nucleic acid. Any other sequence which serves, in some way,to identify a particular probe may alternatively be used as an IDsequence.

The use of barcode sequences as ID sequences is particularly preferred,since this allows all reporter nucleic acid molecules to be detected ina single sequencing reaction. The use of alternative forms of IDsequence, such as unique restriction or primer binding sites, are lessefficient, as they require each combination of restriction enzymes orprimers to be tested in a separate reaction to determine which probeshave interacted to generate reporter nucleic acids. However, there maynonetheless be occasions on which such an alternative type of IDsequence is preferred.

Thus, the reporter nucleic acid molecules (i.e. the extension orligation products) are detected, and said detection comprisesidentification of the ID sequences (preferably barcode sequences) withineach reporter nucleic acid, as detailed above. The detection step notonly comprises detection of the various reporter nucleic acid moleculesgenerated, but also determining the relative amounts of each reporternucleic acid molecule. This may be achieved by any suitable means. Highthroughput DNA sequencing, which as detailed above is a preferred meansof reporter nucleic acid detection, is suitable for relativequantification of reporter nucleic acid molecules, since the number ofeach particular reporter nucleic acid is quantified by the sequencingreaction. As noted above, quantitative PCR is another suitable means bywhich reporter nucleic acids can be detected. Detection of reporternucleic acid molecules by quantitative PCR enables the relative amountsof each reporter nucleic acid to be quantified. Any other suitablemethod for quantifying the relative amounts of each reporter nucleicacid may be used.

Once the reporter nucleic acids have been detected a determination stepis performed, to determine which analytes are present in the sample. Inthis step, firstly the level of background is determined. All reporternucleic acids generated as a result of non-specific probe interactionsmay be deemed background interactions. The relative amount of each ofthese background interactions is determined, such that the level ofbackground interaction is determined. By “non-specific probeinteractions” is meant interactions between probes which are not paired,i.e. interactions between probes which bind different analytes. Suchreporter nucleic acids are extension products and/or ligation productswhich comprise a first ID sequence (e.g. barcode sequence) from a firstproximity probe belonging to a first proximity probe pair and a secondID sequence (e.g. barcode sequence) from a second proximity probebelonging to a second proximity probe pair. Such reporter nucleic acidsmay alternatively by described as extension products and/or ligationproducts which comprise a first ID sequence (e.g. barcode sequence) froma proximity probe specific for a first analyte and a second ID sequence(e.g. barcode sequence) from a proximity probe specific for a second (ordifferent) analyte. As described above, non-specific interactionsbetween unpaired proximity probes may occur between probes free insolution, or when only one probe has bound to its analyte, as a resultof their shared hybridisation sites.

Reporter nucleic acids generated by specific probe interactions are thenanalysed. By “specific probe interactions” is meant interactions betweenprobes within a probe pair, i.e. between two probes which bind to thesame analyte. Such reporter nucleic acids are extension products and/orligation products which comprise a first ID sequence and a second IDsequence (e.g. a first and second barcode sequence) from a proximityprobe pair. Such reporter nucleic acids may alternatively by describedas extension products and/or ligation products which comprise a first IDsequence and a second ID sequence (e.g. a first and second barcodesequence) from proximity probes specific for the same analyte.

Probes within a probe pair may also interact in solution, and soreporter nucleic acids generated by specific probe interactions may alsoconstitute background (i.e. be generated as a result of backgroundinteractions). Therefore the amount of each reporter nucleic acidgenerated by specific probe interactions is compared to the level ofbackground interaction, as determined by the amount of reporter nucleicacids generated as a result of non-specific probe interactions. If areporter nucleic acid generated by a specific probe interaction ispresent at a higher level than the level of background interaction (i.e.the level of non-specific background reporter nucleic acids), thisindicates that the analyte bound by the relevant probe pair is presentin the sample. On the other hand, if a reporter nucleic acid generatedby a specific probe interaction is present at a level which is no higherthan the non-specific background reporter nucleic acids (e.g. if thereporter nucleic acid generated by a specific probe interaction ispresent at a level which is the same or lower than the non-specificbackground reporter nucleic acids), then the interaction between therelevant probe pair is deemed merely to be background. In this case, thefact that the interaction between the probes of the probe pair is merelybackground indicates that the analyte bound by the probe pair is notpresent in the sample.

Alternatively, for any individual target molecule, backgroundinteractions may be defined only as non-specific interactions includinga probe which binds that target molecule. That is to say, for eachtarget molecule background interactions may be defined as non-specificinteractions between a probe which recognises the target molecule and anunpaired probe (i.e. a probe which does not recognise the targetmolecule) which shares its hybridisation site with the probe pair whichrecognises the target molecule. Thus in this case non-specificinteractions between probes, neither of which recognise the targetmolecule, are not considered as background interactions for thatparticular target molecule.

In a particular embodiment, the level of background to which the levelof a specific probe interaction is compared is the average level of thebackground interactions considered, in particular the mean level of thebackground interactions considered.

In a particular embodiment, the first step of the method (i.e. the stepof contacting the sample with a plurality of pairs of proximity probes)further comprises contacting the sample with one or more backgroundprobes which do not bind an analyte, said background probes comprising anucleic acid domain comprising an ID sequence and a hybridisationsequence shared with at least one proximity probe. “Background probes”may also be referred to herein as “inert probes”. As noted above theinert probes do not bind an analyte. Inert probes may nonethelesscomprise an analyte-binding domain, if it is specific for an analytewhich is known not to be present in the sample, in particular anantibody. The inert probe may in effect comprise a “binding domain”which is equivalent to the analyte-binding domain of a functionalproximity probe but which does not perform an analyte-binding function,that is the binding domain equivalent is inert. In one embodiment, theinert domain may be provided by bulk IgG. Alternatively, inert probesmay comprise an inactive analyte-binding domain, i.e. a non-functionalanalyte-binding domain. For instance, inert probes may comprise a shamanalyte-binding domain, such as the constant region of an antibody, orone chain of an antibody (a heavy chain or a light chain only).Alternatively, inert probes may comprise an inert domain, to which thenucleic acid domain is attached but has no function and is not relatedto the analyte-binding domains of the active probes. An inert domain maybe for example a protein which can be added to the assay withoutinterfering with the assay reactions, such as serum albumin (e.g. humanserum albumin or bovine serum albumin). In another alternative, theinert probes are simply nucleic acid molecules, and do not contain anon-nucleic acid domain.

Each inert probe comprises an ID sequence within its nucleic aciddomain. The same type of ID sequence is used in the inert probes as inthe active (i.e. proximity) probes. For instance, if the active probesuse barcode sequences as ID sequences, the inert probes also use barcodesequences as ID sequences. The inert probes each comprise ahybridisation sequence shared with at least one proximity probe.Preferably the inert probes each comprise a hybridisation sequenceshared with multiple proximity probes. When inert probes are used, itmay be that only a single species of inert probe is used, i.e. all inertprobes have the same hybridisation sequence. Preferably however,multiple species of inert probe are used, each inert probe speciescomprising a different hybridisation sequences (shared with a differentproximity probe or different group of proximity probes). It may be thateach different species of inert probe has a different, unique, IDsequence. Alternatively, a common inert probe ID sequence may be used byall inert probes, of all different species. Either way, clearly the IDsequence or sequences used in the inert probes are not shared with anyproximity probe.

Due to the hybridisation sites shared between the inert probes andcertain proximity probes, background interaction in solution betweeninert probes and proximity probes is possible. When an inert probeinteracts with a proximity probe this results in the formation of aduplex between the nucleic acid domains of the two probes. Performanceof the extension and/or ligation reaction results in the formation of anextension and/or ligation product from the duplex formed by the twoprobes. This extension/ligation product is amplified, processed anddetected along with all other products of the assay. Extension/ligationproducts (i.e. reporter nucleic acids) generated from interactionbetween an inert probe and a proximity probe are deemed background inthe determining step.

The multiplex assay used to detect the analytes in the sample ispreferably a PEA. In this embodiment, as noted above, the nucleic aciddomains of each proximity probe pair comprise complementaryhybridisation sequences which hybridise to one another to form theduplex. The duplex formed is subjected to an extension reaction to yieldan extension product. In particular, the extension product of a PEA is alinear extension product.

There are several different variants of PEA, each of which usesproximity probes of slightly different design. The nucleic acid domainsof each proximity probe are designed dependent on the method in whichthe probes are to be used. A representative sample of proximityextension assay formats is shown schematically in FIG. 1 and theseembodiments are described in detail below. In general, in a proximityextension assay, upon binding of a pair of proximity probes to theirtarget analyte the nucleic acid domains of the two probes come intoproximity of each other and interact (i.e. directly or indirectlyhybridise to one another). The interaction between the two nucleic aciddomains yields a nucleic acid duplex comprising at least one free 3′ end(i.e. at least one of the nucleic acid domains within the duplex has a3′ end which can be extended). Addition or activation of a nucleic acidpolymerase enzyme within the assay mix leads to extension of the atleast one free 3′ end. Thus at least one of the nucleic acid domainswithin the duplex is extended, using its paired nucleic acid domain astemplate. The extension product obtained comprises ID sequences whichindicate from which two probes the extension product was generated.

The nucleic acid domains of proximity probes may be single or partiallydouble-stranded. The nucleic acid domains may hybridise to one another,and one domain may template the extension of the other domain. One orboth domains may be extended. Where a nucleic acid domain is partiallydouble-stranded, single-stranded portions of the domains may hybridiseto one another. The single-stranded portions may thus be at the 3′ endof the strand. Where a nucleic acid domain is partially double-stranded,one strand may be conjugated to the analyte binding domain, and theother strand may be hybridised to the conjugated strand. In particularembodiments, the single-stranded portion of a partially double-strandeddomain may be part of the strand which is hybridised to the conjugatedstrand. As will be described in more detail below, the hybridised strand(as opposed to the conjugated strand) of a partially double-strandednucleic acid domain may be viewed as a “splint strand” or splintoligonucleotide.

Version 1 of FIG. 1 depicts a “conventional” proximity extension assay,wherein the nucleic acid domain (shown as an arrow) of each proximityprobe is attached to the analyte-binding domain (shown as an inverted“Y”) by its 5′ end, thereby leaving two free 3′ ends. When saidproximity probes bind to their respective analyte (the analyte is notshown in the figure) the nucleic acid domains of the probes, which arecomplementary at their 3′ ends, are able to interact by hybridisation,i.e. to form a duplex. The addition or activation of a nucleic acidpolymerase enzyme in the assay mixture allows each nucleic acid domainto be extended using the nucleic acid domain of the other proximityprobe as template, yielding an extension product.

Version 2 of FIG. 1 depicts an alternative proximity extension assay,wherein the nucleic acid domain of the first proximity probe is attachedto the analyte-binding domain by its 5′ end and the nucleic acid domainof the second proximity probe is attached to the analyte-binding domainby its 3′ end. The nucleic acid domain of the second proximity probetherefore has a free 5′ end (shown as a blunt arrow), which cannot beextended using a typical nucleic acid polymerase enzyme (which extendonly 3′ ends). The 3′ end of the second proximity probe is effectively“blocked”, i.e. it is not “free” and it cannot be extended because it isconjugated to, and therefore blocked by, the analyte-binding domain. Inthis embodiment, when the proximity probes bind to their respectiveanalyte-binding targets on the analyte, the nucleic acid domains of theprobes, which share a region of complementarity at their 3′ ends, areable to interact by hybridisation, i.e. form a duplex. However, incontrast to version 1, only the nucleic acid domain of the firstproximity probe (which has a free 3′ end) may be extended using thenucleic acid domain of the second proximity probe as a template,yielding an extension product.

In version 3 of FIG. 1 , like version 2, the nucleic acid domain of thefirst proximity probe is attached to the analyte-binding domain by its5′ end and the nucleic acid domain of the second proximity probe isattached to the analyte-binding domain by its 3′ end. The nucleic aciddomain of the second proximity probe therefore has a free 5′ end (shownas a blunt arrow), which cannot be extended. However, in thisembodiment, the nucleic acid domains which are attached to the analytebinding domains of the respective proximity probes do not have regionsof complementarity and therefore are unable to form a duplex directly.Instead, a third nucleic acid molecule is provided that has a region ofhomology with the nucleic acid domain of each proximity probe. Thisthird nucleic acid molecule acts as a “molecular bridge” or a “splint”between the nucleic acid domains. The splint oligonucleotide bridges thegap between the nucleic acid domains, allowing them to interact witheach other indirectly, i.e. each nucleic acid domain forms a duplex withthe splint oligonucleotide.

Thus, when the proximity probes bind to their respective analyte-bindingtargets on the analyte, the nucleic acid domains of the probes eachinteract by hybridisation, i.e. form a duplex, with the splintoligonucleotide. It can be seen therefore that the third nucleic acidmolecule or splint may be regarded as the second strand of a partiallydouble-stranded nucleic acid domain provided on one of the proximityprobes. For example, one of the proximity probes may be provided with apartially double-stranded nucleic acid domain, which is attached to theanalyte binding domain via the 3′ end of one strand and in which theother (non-attached) strand has a free 3′ end. Thus such a nucleic aciddomain has a terminal single-stranded region with a free 3′ end. In thisembodiment the nucleic acid domain of the first proximity probe (whichhas a free 3′ end) may be extended using the “splint oligonucleotide”(or single stranded 3′ terminal region of the other nucleic acid domain)as a template. Alternatively or additionally, the free 3′ end of thesplint oligonucleotide (i.e. the unattached strand, or the 3′single-stranded region) may be extended using the nucleic acid domain ofthe first proximity probe as a template.

As is apparent from the above description, in one embodiment, the splintoligonucleotide may be provided as a separate component of the assay. Inother words it may be added separately to the reaction mix (i.e. addedseparately to the proximity probes to the sample containing theanalytes). Notwithstanding this, since it hybridises to a nucleic acidmolecule which is part of a proximity probe, and will do so upon contactwith such a nucleic acid molecule, it may nonetheless be regarded as astrand of a partially double-stranded nucleic acid domain, albeit thatit is added separately. Alternatively, the splint may be pre-hybridisedto one of the nucleic acid domains of the proximity probes, i.e.hybridised prior to contacting the proximity probe with the sample. Inthis embodiment, the splint oligonucleotide can be seen directly as partof the nucleic acid domain of the proximity probe, i.e. wherein thenucleic acid domain is a partially double-stranded nucleic acidmolecule, e.g. the proximity probe may be made by linking adouble-stranded nucleic acid molecule to an analyte-binding domain(preferably the nucleic acid domain is conjugated to the analyte-bindingdomain by a single strand) and modifying said nucleic acid molecule togenerate a partially double-stranded nucleic acid domain (with asingle-stranded overhang capable of hybridising to the nucleic aciddomain of the other proximity probe).

Hence, the extension of the nucleic acid domain of the proximity probesas defined herein encompasses also the extension of the “splint”oligonucleotide. Advantageously, when the extension product arises fromextension of the splint oligonucleotide, the resultant extended nucleicacid strand is coupled to the proximity probe pair only by theinteraction between the two strands of the nucleic acid molecule (byhybridisation between the two nucleic acid strands). Hence, in theseembodiments, the extension product may be dissociated from the proximityprobe pair using denaturing conditions, e.g. increasing the temperature,decreasing the salt concentration etc.

Whilst the splint oligonucleotide depicted in Version 3 of FIG. 1 isshown as being complementary to the full length of the nucleic aciddomain of the second proximity probe, this is merely an example and itis sufficient for the splint to be capable of forming a duplex with theends (or near the ends) of the nucleic acid domains of the proximityprobes, i.e. to form a bridge between the nucleic acid domains of thetwo probes.

In another embodiment, the splint oligonucleotide may be provided as thenucleic acid domain of a third proximity probe as described in WO2007/107743, which is incorporated herein by reference, whichdemonstrates that this can further improve the sensitivity andspecificity of proximity probe assays.

Version 4 of FIG. 1 is a modification of Version 1, wherein the nucleicacid domain of the first proximity probe comprises at its 3′ end asequence that is not fully complementary to the nucleic acid domain ofthe second proximity probe. Thus, when said proximity probes bind totheir respective analyte the nucleic acid domains of the probes are ableto interact by hybridisation, i.e. to form a duplex, but the extreme 3′end of the nucleic acid domain (the part of the nucleic acid moleculecomprising the free 3′ hydroxyl group) of the first proximity probe isunable to hybridise to the nucleic acid domain of the second proximityprobe and therefore exists as a single stranded, unhybridised, “flap”.On the addition or activation of a nucleic acid polymerase enzyme, onlythe nucleic acid domain of the second proximity probe may be extendedusing the nucleic acid domain of the first proximity probe as template.Thus in this embodiment, only the 3′ end of the nucleic acid domain ofthe second proximity probe is “free”—the 3′ end of the nucleic aciddomain of the first proximity probe is not “free”, because it is notcomplementary to the nucleic acid domain of the second proximity probe,and thus is not hybridised to it and cannot be extended.

Version 5 of FIG. 1 could be viewed as a modification of Version 3.However, in contrast to Version 3, the nucleic acid domains of bothproximity probes are attached to their respective analyte-bindingdomains by their 5′ ends. In this embodiment the 3′ ends of the nucleicacid domains are not complementary and hence the nucleic acid domains ofthe proximity probes cannot interact or form a duplex directly. Instead,a third nucleic acid molecule is provided that has a region of homologywith the nucleic acid domain of each proximity probe. This third nucleicacid molecule acts as a “molecular bridge” or a “splint” between thenucleic acid domains. This “splint” oligonucleotide bridges the gapbetween the nucleic acid domains, allowing them to interact with eachother indirectly, i.e. each nucleic acid domain forms a duplex with thesplint oligonucleotide. Thus, when the proximity probes bind to theirrespective analyte, the nucleic acid domains of the probes each interactby hybridisation, i.e. form a duplex, with the splint oligonucleotide.

In accordance with Version 3, it can be seen therefore that the thirdnucleic acid molecule or splint may be regarded as the second strand ofa partially double stranded nucleic domain provided on one of theproximity probes. In a preferred example, one of the proximity probesmay be provided with a partially double-stranded nucleic acid domain,which is attached to the analyte binding domain via the 5′ end of onestrand and in which the other (non-attached) strand has a free 3′ end.Thus such a nucleic acid domain has a terminal single stranded regionwith at least one free 3′ end. In this embodiment the nucleic aciddomain of the second proximity probe (which has a free 3′ end) may beextended using the “splint oligonucleotide” as a template. Alternativelyor additionally, the free 3′ end of the splint oligonucleotide (i.e. theunattached strand, or the 3′ single-stranded region of the firstproximity probe) may be extended using the nucleic acid domain of thesecond proximity probe as a template.

As discussed above in connection with Version 3, the splintoligonucleotide may be provided as a separate component of the assay. Onthe other hand, since it hybridises to a nucleic acid molecule which ispart of a proximity probe, and will do so upon contact with such anucleic acid molecule, it may be regarded as a strand of a partiallydouble-stranded nucleic acid domain, albeit that it is added separately.Alternatively, the splint may be pre-hybridised to one of the nucleicacid domains of the proximity probes, i.e. hybridised prior tocontacting the proximity probe with the sample. In this embodiment, thesplint oligonucleotide can be seen directly as part of the nucleic aciddomain of the proximity probe, i.e. wherein the nucleic acid domain is apartially double-stranded nucleic acid molecule, e.g. the proximityprobe may be made by linking a double-stranded nucleic acid molecule toan analyte-binding domain (preferably the nucleic acid domain isconjugated to the analyte-binding domain by a single strand) andmodifying said nucleic acid molecule to generate a partiallydouble-stranded nucleic acid domain (with a single-stranded overhangcapable of hybridising to the nucleic acid domain of the other proximityprobe).

Hence, the extension of the nucleic acid domain of the proximity probesas defined herein encompasses also the extension of the “splint”oligonucleotide. Advantageously, when the extension product arises fromextension of the splint oligonucleotide, the resultant extended nucleicacid strand is coupled to the proximity probe pair only by theinteraction between the two strands of the nucleic acid molecule (byhybridisation between the two nucleic acid strands). Hence, in theseembodiments, the extension product may be dissociated from the proximityprobe pair using denaturing conditions, e.g. increasing the temperature,decreasing the salt concentration etc.

Whilst the splint oligonucleotide depicted in Version 5 of FIG. 1 isshown as being complementary to the full length of the nucleic aciddomain of the first proximity probe, this is merely an example and it issufficient for the splint to be capable of forming a duplex with theends (or near the ends) of the nucleic acid domains of the proximityprobes, i.e. to form a bridge between the nucleic acid domains of theproximity probes.

In another embodiment, the splint oligonucleotide may be provided as thenucleic acid domain of a third proximity probe as described in WO2007/107743, which is incorporated herein by reference, whichdemonstrates that this can further improve the sensitivity andspecificity of proximity probe assays.

Version 6 of FIG. 1 is the most preferred embodiment of the presentinvention. As depicted, both probes in a pair are conjugated topartially single-stranded nucleic acid molecules. In each probe, a shortnucleic acid strand is conjugated via its 5′ end to the analyte-bindingdomain. The short nucleic acid strands which are conjugated to theanalyte-binding domains do not hybridise to each other. Rather, eachshort nucleic acid strand is hybridised to a longer nucleic acid strand,which has a single-stranded overhang at its 3′ end (that is to say, the3′ end of the longer nucleic acid strand extends beyond the 5′ end ofthe shorter strand conjugated to the analyte-binding domain. Theoverhangs of the two longer nucleic acid strands hybridise to oneanother, forming a duplex. The longer nucleic acid strands, whichhybridise to each other, are referred to herein as “hybridisationoligonucleotides”. If the 3′ ends of the two longer nucleic acidmolecules hybridise fully to one another, as shown, the duplex comprisestwo free 3′ ends, though the 3′ ends of the longer nucleic acidmolecules may be designed as in Version 4, such that the extreme 3′ endof one of the longer nucleic acid molecules is not complementary to theother, forming a flap, meaning that the duplex contains only one free 3′end.

Thus it can be seen that when the method of the invention is performedusing a PEA, in some embodiments, in each proximity probe pair at leastone nucleic acid domain is partially double-stranded. It may be that ineach proximity probe pair one nucleic acid domain is partiallydouble-stranded (as in Versions 3 and 5). It is preferred that in eachproximity probe pair both nucleic acid domains are partiallydouble-stranded, as in Version 6.

As detailed in respect of Version 6, it is preferred that the partiallydouble-stranded nucleic acid domain comprises:

(i) a first oligonucleotide conjugated to the analyte-binding domain;and

(ii) a hybridisation oligonucleotide comprising the first hybridisationsequence, the ID sequence and a second hybridisation sequence, the firsthybridisation sequence being located at the 3′ end of the hybridisationoligonucleotide;

wherein the double-stranded part of the nucleic acid domain comprises aduplex between the second hybridisation sequence of the hybridisationoligonucleotide and the first oligonucleotide, and the single-strandedpart of the nucleic acid domain comprises the first hybridisationsequence of the hybridisation oligonucleotide.

In a particular embodiment, the hybridisation oligonucleotide comprises,from 5′ to 3′, the second hybridisation sequence, the ID sequence(preferably barcode sequence) and the first hybridisation sequence, andthe ID sequence (preferably barcode sequence) is located in thesingle-stranded part of the nucleic acid domain.

It may be the case that all proximity probes comprise the same firstoligonucleotide and the same second hybridisation sequence (within thehybridisation oligonucleotide). In other words, all proximity probes mayshare a universal first oligonucleotide and second hybridisationsequence. This may result in a more straightforward manufacturingprocess for the probes.

As detailed above, the first oligonucleotide and the secondhybridisation sequence are complementary to one another, such that thetwo sequences are capable of hybridising to each other. In a particularembodiment the second hybridisation site is complementary to theentirety of the first oligonucleotide, such that the duplex formedbetween them comprises the entire first oligonucleotide. This is notessential though, and it may be that the second hybridisation site iscomplementary only to part of the first oligonucleotide, such that theformed between them comprises only part of the first oligonucleotide. Asalso noted above, the first hybridisation sequence is located at the 3′end of the hybridisation oligonucleotide, such that when two probes withcomplementary first hybridisation sequences come into proximity, the 3′ends of their hybridisation oligonucleotides hybridise to one another.By “located at the 3′ end” may mean that the first hybridisationsequence extends to the 3′ terminus of each hybridisationoligonucleotide, i.e. the first hybridisation sequences may include the3′ nucleotide of each hybridisation oligonucleotide. However, this isnot essential, and the first hybridisation sequence may alternativelyextend only to the 3′ terminus of one hybridisation oligonucleotide ineach probe pair. The nucleic acid domains used in PEA Version 6 abovemay thus be designed in the same manner as those of Version 4, such thatone of the hybridisation oligonucleotides in each probe pair comprisesat its 3′ end a sequence that is not fully complementary to thehybridisation oligonucleotide of the other proximity probe, and thusforms a single stranded, unhybridised, “flap”.

Thus, following hybridisation of two nucleic acid domains to each other,at least one hybridisation oligonucleotide is extended to generate theextension product (in other words, one or both hybridisationoligonucleotides are extended to generate the extension product). If thefirst hybridisation sequences extend to the 3′ termini of bothhybridisation oligonucleotides in each probe pair, it may be that bothhybridisation oligonucleotides are extended to generate the extensionproduct. On the other hand, if one of the hybridisation oligonucleotidescomprises an unhybridised flap at its 3′ end (as detailed above), onlyone of the hybridisation oligonucleotides is extended to generate theextension product (i.e. the hybridisation oligonucleotide without theflap).

When the multiplex assay performed is a PEA, it is preferred that theextension reaction is performed in the context of a PCR amplification,or in other words a single reaction, including a PCR amplification, isperformed to achieve both extension of the proximity probe nucleic aciddomains, thus generating the reporter nucleic acid molecule, andamplification of the generated reporter nucleic acid molecule. In thisembodiment, rather than beginning with a denaturation step (as isnormally the case in PCR), the reaction begins with an extension step,during which the reporter nucleic acid molecule is generated.Thereafter, a standard PCR is performed to amplify the reporter nucleicacid molecule, beginning with denaturation of the reporter molecule. Asdetailed above, the PCR is preferably performed using common primerswhich bind to common sequences at the ends of the reporter nucleic acidmolecule. As detailed below, one or both of the primers may alsocomprise a sequencing adapter. In other words, the extension andamplification steps of the method of the invention may be performed in asingle reaction.

In another embodiment the multiplex assay used to detect the analytes inthe sample is a PLA. The PLA used may be a “standard” PLA. By this ismeant a PLA using a single splint oligonucleotide to join the nucleicacid domains of two proximity probes. In a standard PLA, the nucleicacid domains of each proximity probe pair comprise paired hybridisationsequences which hybridise to the splint oligonucleotide to form theduplex. The nucleic acid domains of the proximity probe pair areconjugated to their respective probes such that in each pair, oneproximity probe has a nucleic acid domain with a free 3′ end and theother has a nucleic acid domain with a free 5′ end, such that the freeends of the nucleic acid domains of the two probes can be ligatedtogether. The splint oligonucleotide may comprise an extension blockerat its 3′ end, such that it cannot be extended. Following duplexformation the nucleic acid domains of the two proximity probes aredirectly or indirectly ligated to each other to generate a ligationproduct comprising the ID sequence of the first proximity probe and theID sequence of the second proximity probe.

When the multiplex assay is a PLA, it is preferred that the nucleic aciddomains of the proximity probe pair hybridise to the splintoligonucleotide such that there is a break in the duplex between the twonucleic acid domains, but no gap. In other words, the 3′ terminus of onenucleic acid domain may hybridise to the nucleotide of the splintdirectly adjacent to the nucleotide of the splint to which the 5′terminus of the other nucleic acid domain hybridises. This enablesdirect ligation of the two nucleic acid domains to each other.

Alternatively, the nucleic acid domains of the proximity probe pair mayhybridise to the splint oligonucleotide such that there is a gap betweenthe 3′ terminus of one nucleic acid domain and the 5′ terminus of theother nucleic acid domain. In this embodiment, the duplex formed betweenthe splint oligonucleotide and two probe nucleic acid domains comprisesa length of single-stranded nucleic acid from the splintoligonucleotide, separating the two parts of the duplex. Thesingle-stranded gap may be any number of nucleotides in length. In thisembodiment, a gap-filling extension reaction is performed to fill thegap between the ends of the two probe nucleic acid domains (i.e. theprobe nucleic acid domain comprising the free 3′ end is extended, inorder to fill the gap). Following gap-filling, the nucleic acid domainsof the two splint oligonucleotides are ligated to each other using aligase enzyme. Ligation of nucleic acid domains to each other followinggap-filling is referred to herein as “indirect ligation” of the nucleicacid domains to each other.

The gap-filling extension reaction is performed using a polymeraseenzyme lacking strand displacement activity, such that extension endswhen the gap is filled, rather than displacing the hybridised nucleicacid domain downstream of the free 3′ end. Non-displacing polymerasesinclude the T4 DNA polymerase. Other such polymerases are known in theart.

Following ligation, the ligation product is amplified as described above(e.g. by PCR) and detected. These PLA embodiments yield a linearligation (or extension and ligation) product. It is preferred that aligation product, or an extension and ligation product, generated in themethod of the invention is linear.

Alternatively, the PLA used may be rolling circle amplification PLA(PLA-RCA). This PLA format uses two splint oligonucleotides which areligated to each other to generate a ligation product. PLA-RCA isdescribed in e.g. Söderberg et al., Nature Methods 3(12): 995-1000(2006). In this embodiment, the proximity probes comprise nucleic aciddomains each comprising two hybridisation sequences. The firsthybridisation sequence is complementary to a hybridisation sequence on afirst splint oligonucleotide, and the second hybridisation sequence iscomplementary to a hybridisation sequence on a second splintoligonucleotide. The first hybridisation sequences are paired, asdescribed above, such that the same pair of first hybridisationsequences is shared by a number of proximity probe pairs. Thesehybridise to a particular first splint oligonucleotide, as detailedabove. The second hybridisation sequences may also be paired, but morepreferably these are universal sites, shared by all proximity probenucleic acid domains in the multiplex assay, such that only a singlesecond splint oligonucleotide is required for all proximity probe pairs.

In PLA-RCA, the ID sequences (preferably barcode sequences) of the probenucleic acid domains are located between the first and secondhybridisation sites. Upon binding of the two splint oligonucleotides tothe probe nucleic acid domains, a gap-filling extension reaction isperformed, as described above. The two splint oligonucleotides are thenligated to each other to form a circular molecule, which is amplified byrolling circle amplification, and detected.

As noted above, it is preferred that the reporter nucleic acid isdetected by massively parallel DNA sequencing, and that this generallyrequires the addition of sequencing adapters to the DNA molecule to besequenced. As detailed above, sequencing adapters function to immobilisethe DNA molecule on a surface.

The method of the invention may thus include the addition of one or moreadapters for sequencing (sequencing adapters) to the reporter nucleicacids.

Commonly, the sequencing adapters are nucleic acid molecules (inparticular DNA molecules). In this instance, short oligonucleotidescomplementary to the adapter sequences are conjugated to theimmobilisation surface (e.g. the surface of a bead or flow cell) toenable annealing of the target DNA molecules to the surface, via theadapter sequences. Alternatively, any other pair of binding partners maybe used to conjugate the target DNA molecule to the immobilisationsurface, e.g. biotin and avidin/streptavidin. In this case biotin may beused as the sequencing adapter, and avidin or streptavidin conjugated tothe immobilisation surface to bind the biotin sequencing adapter, orvice versa.

Sequencing adapters may thus be short oligonucleotides (preferably DNA),generally 10-30 nucleotides long (e.g. 15-25 or 20-25 nucleotides long).As detailed above, the purpose of a sequencing adapter is to enableannealing of the target DNA molecules to an immobilisation surface, andaccordingly the nucleotide sequence of a nucleic acid adaptor isdetermined by the sequence of its binding partner conjugated to theimmobilisation surface. Aside from this, there is no particularconstraint on the nucleotide sequence of a nucleic acid sequencingadaptor.

A sequencing adapter may be added to a reporter nucleic acid of theinvention during PCR amplification. In the case of a nucleic acidsequencing adapter this can be achieved by including a sequencingadapter nucleotide within in one or both primers. Alternatively, if thesequencing adaptor is a non-nucleic acid sequencing adaptor (e.g. aprotein/peptide or small molecule) an adapter may be conjugated to oneor both PCR primers. Alternatively, a sequencing adapter may be attachedto a reporter nucleic acid molecule by directly ligating or conjugatingthe sequencing adapter to the reporter nucleic acid molecule. Preferablythe one or more sequencing adapters used in the present method arenucleic acid sequencing adapters.

One or more nucleic acid sequencing adapters may thus be added to thereporter nucleic acid in one or more ligation and/or amplificationsteps. Thus if, for instance, two sequencing adapters are added to thereporter nucleic acid molecule (one at each end), these may be added ina single step (e.g. by PCR amplification using a pair of primers whichboth contain a sequencing adapter) or in two steps. The two steps may beperformed using the same or different methods, e.g. a first sequencingadapter may be added to the reporter nucleic acid molecule by ligationand the second by PCR amplification, or vice versa; or a firstamplification reaction may be performed to add a first sequencingadapter to the reporter nucleic acid molecule, followed by a secondamplification reaction to add a second sequencing adapter to thereporter nucleic acid molecule.

As noted above, one or more sequencing adapters may be added to thereporter nucleic acid molecule. By this is meant one or two sequencingadapters—since sequencing adapters are added to the ends of a DNAmolecule, the maximum number of sequencing adapters which can be addedto a single DNA molecule (e.g. reporter nucleic acid) is two. Thus asingle sequencing adapter may be added to one end of a reporter nucleicacid molecule, or two sequencing adapters may be added to a reporternucleic acid molecule, one to each end. In a particular embodiment theIllumina P5 and P7 adapters are used, i.e. the P5 adapter is added toone end of the reporter nucleic acid molecule and the P7 adapter isadded to the other end. The sequence of the P5 adapter is set forth inSEQ ID NO: 1 (AAT GAT ACG GCG ACC ACC GA) and the sequence of the P7adapter is set forth in SEQ ID NO: 2 (CAA GCA GAA GAC GGC ATA CGA GAT).

PCR amplification may thus be combined with addition of one or moresequencing adapters to the reporter nucleic acid molecule. This may beachieved by amplification of the reporter nucleic acid molecule using aprimer pair comprising at least one sequencing adapter. In thisinstance, at least one primer in the primer pair comprises a sequencingadapter upstream of the sequence which binds the reporter nucleic acidmolecule. Thus the sequencing adapter is generally located at the 5′ endof any primer within which it is contained.

In a particular embodiment, an amplification step is performed using aprimer pair comprising one primer which includes a sequencing adapter,such that a single sequencing adapter is added to one end of thereporter nucleic acid molecule.

In another embodiment, an amplification step is performed using a primerpair in which both primers comprise a sequencing adapter, such that asequencing adapter is added to each end of the reporter nucleic acidmolecule in a single amplification step.

In another embodiment, two separate amplification reactions areperformed to add a sequencing adaptor to each end of the reporternucleic acid molecule, wherein each amplification step adds a differentsequencing adaptor to a different end of the molecule.

In another embodiment, an initial amplification step is performed usingprimers which do not comprise sequencing adapters. The amplifiedreporter nucleic acid molecules are then subjected to one or morefurther amplification reactions to add sequencing adapters to each endof the molecule, as described above.

It is preferred that the reporter nucleic acid (i.e. extension and/orligation product) is amplified in two PCR steps. In the first PCRreaction a first sequencing adapter is added to one end of the extensionproduct or ligation product. The product of the first PCR reaction isthen amplified in a second PCR reaction, in which a second sequencingadapter is added to the other end of the reporter nucleic acid. In aparticular embodiment, the first PCR reaction is performed with anucleic acid polymerase that also has 3′ to 5′ exonuclease activity, andthe second PCR reaction is performed with a nucleic acid polymerase thatlacks 3′ to 5′ exonuclease activity, as described in WO 2012/104261.Suitable nucleic acid polymerases which have 3′ to 5′ exonucleaseactivity include T4 DNA polymerase, T7 DNA polymerase, Phi29 (ϕ29) DNApolymerase, DNA polymerase I, Klenow fragment of DNA polymerase I,Pyrococcus furiosus (Pfu) DNA polymerase and Pyrococcus woesei (Pwo) DNApolymerase. Suitable nucleic acid polymerases which lack 3′ to 5′exonuclease activity include the α subunit of DNA polymerase III, theKlenow exo(−) fragment of DNA polymerase I, Taq polymerase, Pfu (exo⁻)DNA polymerase and Pwo (exo⁻) DNA polymerase.

In another embodiment, the same polymerase may be used for both PCRsteps, e.g. Pwo or Pfu polymerase.

The method of the present invention may be used to assay multiplesamples simultaneously. In this case, separate multiplex assays areperformed as described above for each sample. Once the extension and/orligation product has been generated, a sample index sequence is added. Asample index is a nucleotide sequence which identifies the source samplefrom which an extension and/or ligation product is derived. Thus adifferent nucleotide sequence is used as the sample index sequence forextension/ligation products derived from each different sample.Conversely, all extension and/or ligation products from a particularsample are labelled with the same sample index sequence.

Once all products have been labelled with a sample index, the productsof multiple samples may be pooled and analysed together. When thereporter nucleic acids are sequenced, the sample index will indicatewhich sample each individual reporter nucleic acid molecule is from. Anynucleotide sequence may be used as the sample index. Sample indexsequences may be of any length but are preferably relatively short, e.g.3-12, 4-10 or 4-8 nucleotides.

The sample index sequence may be added to the extension/ligationproducts by any suitable method, for instance the sample index may beadded in an amplification reaction (e.g. by PCR) or in a ligationreaction. Notably, if the reporter nucleic acid molecules are to beanalysed by massively parallel DNA sequencing, and require sequencingadapters at both ends, the sample index sequence cannot be added suchthat it is, ultimately, located at an end of the reporter nucleic acidmolecules.

In a preferred embodiment, sample index sequences are added to theextension/ligation products during PCR amplification. As noted above,sequencing adapters may also be added to the extension/ligation productsduring PCR amplification. In a particular embodiment, a dedicatedamplification step may be performed exclusively to add the sample indexto the reporter nucleic acid molecule. In another embodiment, the sampleindex may be added during PCR amplification at the same time as one ormore sequencing adapters. For instance, if a single PCR amplification isperformed to add sequencing adapters to both ends of the reporternucleic acid molecules, a sample index may be added at the same time.Alternatively, if sequencing adapters are added to each end of thereporter nucleic acid molecule in two sequential PCR amplifications, asample index may be added during either PCR amplification. The sampleindex may be added during the first PCR amplification, in which case itmay be added to the reporter nucleic acid at the same end as thesequencing adapter is added (internal to the sequencing adapter) or atthe opposite end. Alternatively, the sample index may be added duringthe second PCR amplification, in which case it must be added to thereporter nucleic acid at the same end the sequencing adapter is added(internal to the sequencing adapter. Alternatively, a ligation step maybe performed to add the sample index to the end of each reporter nucleicacid molecule prior to amplification.

The second aspect of the present invention is a product which may beused to perform the method of the first aspect of the invention. Inparticular, as set out above, the product comprises:

(i) a plurality of proximity probe pairs, wherein each proximity probepair comprises a first proximity probe and a second proximity probe, andeach proximity probe comprises:

-   -   (a) a protein-binding domain specific for a protein; and    -   (b) a nucleic acid domain,

wherein both probes within each pair comprise protein-binding domainsspecific for the same protein, and can simultaneously bind to theprotein; and each probe pair is specific for a different protein;

wherein the nucleic acid domain of each proximity probe comprises an IDsequence and at least a first hybridisation sequence, wherein the IDsequence of each proximity probe is different; and wherein in eachproximity probe pair, the first proximity probe and the second proximityprobe comprise paired hybridisation sequences; and, optionally

(ii) a plurality of splint oligonucleotides, each splint oligonucleotidecomprising hybridisation sequences complementary to each of the pairedhybridisation sequences of a proximity probe pair;

wherein the hybridisation sequences of each proximity probe pair areconfigured such that upon binding of the first and second proximityprobe to their protein, the respective paired hybridisation sequences ofthe first and second proximity probes hybridise to each other or to asplint oligonucleotide;

and wherein at least one pair of hybridisation sequences is shared by atleast two pairs of proximity probes.

The various features of this aspect are the same as the equivalentfeatures of the first aspect (e.g. the proximity probes, ID sequences,splint oligonucleotides, hybridisation sequences, etc.). Notably, inthis aspect of the invention, both probes within each probe paircomprise protein-binding domains specific for the same protein. In otherwords, in each probe pair of the product, both probes bind the sameprotein. As detailed above, the two probes in each probe pair bind theirtarget protein at different epitopes, such that they do not interferewith each other's binding to the target. The probes of the invention maybe designed for use in any style or variant of PEA or PLA as describedabove.

In an embodiment, the product of the invention further comprises one ormore background probes (or inert probes) which do not bind an analyte,as described above. As in the method of the invention, it is preferredthat a significant proportion of probe pairs share their hybridisationsequences with at least one other proximity probe pair. In particularembodiments, at least 25%, 50% or 75% of proximity probe pairs sharetheir hybridisation sequences with another proximity probe pair (i.e.with at least one other proximity probe pair), as in the method. In aparticular embodiment, all proximity probe pairs share theirhybridisation sequences with at least one other proximity probe pair.However, as is apparent from the above, in another embodiment at leastone pair of hybridisation sequences is unique to a single pair ofproximity probes. That is to say, at least one pair of proximity probesdoes not share its hybridisation sequences with any other proximityprobe pair. In particular embodiments, up to 75%, 50% or 25% of pairs ofproximity probes do not share their hybridisation sequences with anyother proximity probe pair. As in the method, in certain embodiments nomore than 20, 15, 10 or 5 proximity probe pairs in the product share thesame pair of hybridisation sequences.

The product of the invention may be provided as a single compositioncomprising all proximity probes (and if present, splint oligonucleotidesand/or inert probes). Alternatively, all components of the product maybe provided in separate containers. For instance, proximity probe pairs,splint oligonucleotides and inert probes may all be provided in separatecontainers. If desired, each probe pair, or even each individualproximity probe, may be provided in a separate container, as may eachdifferent splint oligonucleotide and each different inert probe.

The product may further comprise additional components for use in themethod of the invention. For instance, the product may comprise one ormore nucleic acid polymerase enzymes for use in the extension and/oramplification steps, and/or a ligase enzyme if a ligation step isrequired. The product may comprise primers for use in amplification. Asdetailed above, the primers used in the amplification step(s) maycomprise sequence adapters for nucleic acid sequencing and/or sampleindex sequences. The product may also comprise nucleotides (e.g. dATP,dCTP, dGTP and dTTP) for use in the extension/amplification reactions.The product may comprise a solid base to which the reporter nucleicacids can be immobilised for sequencing, e.g. a flow cell or bead.

The invention may be further understood by reference to the non-limitingexamples below, and the figures.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a schematic representation of six different versions ofproximity extension assays, described in detail above. The inverted ‘Y’shapes represent antibodies, as an exemplary proximity probeanalyte-binding domain.

FIG. 2 shows a comparison of results obtained for the level ofexpression of four analytes across six different samples, as determinedby multiplex PEA, using either a traditional negative control or ashared hybridisation site negative control.

FIG. 3 shows a comparison of results obtained for the level ofexpression of five analytes across six different samples (same samplesas used for FIG. 2 ), as determined by multiplex PEA, using either atraditional negative control or a shared hybridisation site negativecontrol.

EXAMPLES

Plasma samples were obtained from 6 donors: 3 healthy subjects, onesubject diagnosed with breast cancer, one diagnosed with rheumatoidarthritis (RA) and one diagnosed with inflammatory bowel disease (IBD).

A multiplex PEA was performed (using probes comprising antibodiesconjugated to nucleic acid domains having the structure described inVersion 6, above) to detect 9 proteins in the samples: NPDC1 (UniProtQ9NQX5); AHCY (UniProt P23526); TM (UniProt P07204); ANGPTL1 (UniProtO95841); LOX-1 (UniProt P78380); SEMA3F (UniProt Q13275); CDH2 (UniProtP19022); CANT1 (UniProt Q8WVQ1); and CA13 (UniProt Q8N1Q1). The probestargeted against NPDC1, AHOY, TM and ANGPTL1 all shared a pair ofhybridisation sites; and the probes targeted against LOX-1, SEMA3F,CDH2, CANT1 and CA13 all shared a different pair of hybridisation sites.Each probe contained a unique barcode sequence. A negative control wasalso used, comprising phosphate buffered saline with 1% bovine serumalbumin without sample.

The PEA was performed as described above. During amplification of theextension products, P5 and P7 sequencing adapters were added to each endof the products, along with a unique sample index for reporter nucleicacids from each different sample, and all extension products sequencedby massively parallel DNA sequencing, employing reversible dyeterminator sequencing technique using an Illumina NovaSeq platform.

Background from standard negative control for a target was determinedfrom the paired barcode interaction of probes for the target. Backgroundfrom shared hybridisation sites for a target was determined from themean value of the mismatched interactions (as determined by mismatchedbarcodes) between each respective probe of the pair of probes for thetarget and other probes within the group (i.e. probes that sharehybridisation sites with the probes for the target), for each sample. Inother words, for each target background from shared hybridisation siteswas defined as non-specific interactions between each probe for thetarget and other probes having shared hybridisation sites. Non-specificinteractions between probes, neither of which bind the target, were notincluded in the calculation of background.

The following results were obtained from the two groups of targetanalytes:

Group 1—Linear Analysis

Signal above background from negative control:

NPDC1 AHCY TM ANGPTL1 IBD Subject 4.744595 18.77055 24.26055 5.650493 RASubject 24.15997 23.02676 31.97209 16.44529 Breast Cancer Subject15.63025 5.763718 21.46488 16.13142 Healthy Control 1 10.11761 9.27304918.64168 24.23299 Healthy Control 2 14.58207 2.398616 26.94637 17.11784Healthy Control 3 26.35638 6.522163 38.96036 24.39238

Signal above background from shared hybridisation sites:

NPDC1 AHCY TM ANGPTL1 IBD Subject 6.587558 24.20433 33.56129 6.621035 RASubject 30.17537 30.42173 36.67241 14.92188 Breast Cancer Subject18.81776 6.457732 29.32031 14.772 Healthy Control 1 12.29969 10.4435121.19694 21.6378 Healthy Control 2 14.15044 2.597143 28.14347 13.73367Healthy Control 3 31.26646 7.661677 46.84286 21.24901

Group 1—Logarithmic Analysis (Base 2)

Signal above background from negative control:

NPDC1 AHCY TM ANGPTL1 IBD Subject 2.246285 4.230399 4.60054 2.498377 RASubject 4.594547 4.52524 4.998741 4.039603 Breast Cancer Subject3.966269 2.527 4.423906 4.011802 Healthy Control 1 3.338797 3.2130444.22046 4.598901 Healthy Control 2 3.866123 1.262202 4.752019 4.097428Healthy Control 3 4.72008 2.70535 5.283935 4.608359

Signal above background from shared hybridisation sites:

NPDC1 AHCY TM ANGPTL1 IBD Subject 2.719744 4.597194 5.068726 2.727057 RASubject 4.915299 4.92703 5.196623 3.899357 Breast Cancer Subject4.234023 2.691028 4.873829 3.884793 Healthy Control 1 3.62055 3.3845354.405784 4.435482 Healthy Control 2 3.822775 1.376925 4.814728 3.779645Healthy Control 3 4.966544 2.93766 5.549757 4.409324

The logarithmic results are shown in the graph of FIG. 2 .

Group 2—Linear Analysis

Signal above background from negative control:

LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 93.75019 7.710203 11.65482 30.8892122.95267 Subject RA 51.94867 15.51322 13.56623 46.84155 304.8523 SubjectBreast 12.1141 15.45434 16.45051 36.89154 104.631 Cancer Subject Healthy23.56257 8.300925 8.070123 29.4027 4.299637 Control 1 Healthy 18.146796.530255 17.95702 36.27412 14.72176 Control 2 Healthy 25.83432 13.7614413.69109 34.4381 5.858678 Control 3

Signal above background from shared hybridisation sites:

LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 141.2799 18.21138 16.84211 31.7516531.70223 Subject RA 65.79012 31.66038 15.88331 39.08923 338.9779 SubjectBreast 14.1692 29.58474 20.04621 31.2304 117.9876 Cancer Subject Healthy33.38638 17.40281 10.59963 26.28186 5.242938 Control 1 Healthy 23.5920511.80547 21.32377 31.38729 17.40023 Control 2 Healthy 32.05737 26.647816.27754 27.05954 6.934537 Control 3

Group 2—Logarithmic Analysis (Base 2)

Signal above background from negative control:

LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 6.55075 2.946769 3.542855 4.9490314.52059 Subject RA 5.699015 3.955426 3.761948 5.549717 8.251967 SubjectBreast 3.598615 3.94994 4.04006 5.205218 6.709166 Cancer Subject Healthy4.558425 3.053272 3.012591 4.877877 2.104215 Control 1 Healthy 4.1816432.707139 4.166476 5.180869 3.879879 Control 2 Healthy 4.691217 3.7825593.775165 5.105934 2.550575 Control 3

Signal above background from shared hybridisation sites:

LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 7.142412 4.186769 4.074001 4.9887594.986513 Subject RA 6.039799 4.984607 3.989439 5.288699 8.405047 SubjectBreast 3.824686 4.886781 4.325258 4.964879 6.882491 Cancer SubjectHealthy 5.061188 4.121249 3.405942 4.715995 2.390375 Control 1 Healthy4.560229 3.561384 4.41439 4.972109 4.121035 Control 2 Healthy 5.0025844.735944 4.024811 4.758066 2.7938 Control 3

The logarithmic results are shown in the graph of FIG. 3 .

While for both groups of analytes, the actual values of the signalsabove background may differ between the two types of control (negativecontrol and shared hybridisation sites control), the values shiftapproximately equally for each analyte for each sample (i.e. there is aparallel shift). This is demonstrated by the high R² values for theresults obtained with each analyte, which indicate a very highcorrelation between the degree of signal above background as determinedby each of the two methods. These results demonstrate that the use ofshared hybridisation sites is a valid alternative to a standard negativecontrol since the relative signal levels of an analyte is retainedbetween samples. The results from the background determination fromshared hybridisation sites show similar discrimination between samplesas when using a standard negative control.

1. A method for detecting a plurality of analytes in a sample, themethod comprising performing a multiplex proximity-based detectionassay, the assay comprising: (i) contacting the sample with a pluralityof pairs of proximity probes, wherein each proximity probe paircomprises a first proximity probe and a second proximity probe, and eachproximity probe comprises: (a) an analyte-binding domain specific for ananalyte; and (b) a nucleic acid domain, wherein both probes within eachpair comprise analyte-binding domains specific for the same analyte, andcan simultaneously bind to the analyte; and each probe pair is specificfor a different analyte; wherein the nucleic acid domain of eachproximity probe comprises an ID sequence and at least a firsthybridisation sequence, wherein the ID sequence of each proximity probeis different; and wherein: in each proximity probe pair, the firstproximity probe and the second proximity probe comprise pairedhybridisation sequences, such that upon binding of the first and secondproximity probe to their analyte, the respective paired hybridisationsequences of the first and second proximity probes hybridise to eachother or to a common splint oligonucleotide which compriseshybridisation sequences complementary to each of the pairedhybridisation sequences of the first and second proximity probes; andwherein at least one pair of hybridisation sequences is shared by atleast two pairs of proximity probes; (ii) allowing the nucleic aciddomains of the proximity probes to hybridise to one another or to thesplint oligonucleotide, to form a continuous or non-continuous duplexcomprising the hybridisation sequence of a first proximity probe and ahybridisation sequence of a second proximity probe, wherein said duplexcomprises at least one free 3′ end; (iii) subjecting the duplex to anextension and/or ligation reaction to generate an extension and/orligation product which comprises the ID sequence of the first proximityprobe and the ID sequence of the second proximity probe; (iv) amplifyingthe extension product or ligation product; (v) detecting the extensionproduct or ligation product, wherein detection of the extension productor ligation product comprises identification of the ID sequencestherein, and determining the relative amounts of each extension productor ligation product; and (vi) determining which analytes are present inthe sample, wherein: (a) extension products and/or ligation productswhich comprise a first ID sequence from a first proximity probebelonging to a first proximity probe pair and a second ID sequence froma second proximity probe belonging to a second proximity probe pair aredeemed background; and (b) an extension product or ligation productwhich comprises a first ID sequence and a second ID sequence from aproximity probe pair, and which is present in an amount higher than thebackground, indicates that the analyte specifically bound by theproximity probe pair is present in the sample.
 2. The method of claim 1,wherein the analyte is or comprises a protein.
 3. The method of claim 1or 2, wherein the analyte-binding domain is an antibody or fragmentthereof.
 4. The method of any one of claims 1 to 3, wherein step (i)further comprises contacting the sample with one or more backgroundprobes which do not bind an analyte, said background probes comprising anucleic acid domain comprising an ID sequence and a hybridisationsequence shared with at least one proximity probe; wherein an extensionand/or ligation product generated as a result of an interaction betweena background probe and a proximity probe is detected in step (v), anddeemed background in step (vi).
 5. The method of any one of claims 1 to4, wherein the ID sequences are barcode sequences.
 6. The method of anyone of claims 1 to 5, wherein at least one pair of hybridisationsequences is unique to a single pair of proximity probes.
 7. The methodof any one of claims 1 to 6, wherein no more than 10 proximity probepairs share the same pair of hybridisation sequences.
 8. The method ofclaim 7, wherein no more than 5 proximity probe pairs share the samepair of hybridisation sequences.
 9. The method of any one of claims 1 to8, wherein at least 25% of proximity probe pairs share their pair ofhybridisation sequences with another proximity probe pair.
 10. Themethod of claim 9, wherein at least 50% of proximity probe pairs sharetheir pair of hybridisation sequences with another proximity probe pair.11. The method of claim 10, wherein at least 75% of proximity probepairs share their pair of hybridisation sequences with another proximityprobe pair.
 12. The method of any one of claims 1 to 11, wherein theproximity-based detection assay is a proximity extension assay, whereinthe nucleic acid domains of each proximity probe pair comprisecomplementary hybridisation sequences which hybridise to one another toform the duplex; and wherein the duplex is subjected to an extensionreaction, said extension reaction comprising extending the at least onefree 3′ end to generate an extension product comprising the ID sequenceof the first proximity probe and the ID sequence of the second proximityprobe.
 13. The method of any one of claims 1 to 11, wherein theproximity-based detection assay is a proximity ligation assay, whereinthe nucleic acid domains of each proximity probe pair comprise pairedhybridisation sequences which hybridise to the splint oligonucleotide toform the duplex, and wherein step (iii) comprises directly or indirectlyligating the nucleic acid domain of the first proximity probe to thenucleic acid domain of the second proximity probe to generate a ligationproduct comprising the ID sequence of the first proximity probe and theID sequence of the second proximity probe.
 14. The method of claim 12,wherein in each proximity probe pair at least one nucleic acid domain ispartially double-stranded.
 15. The method of claim 14, wherein in eachproximity probe pair both nucleic acid domains are partiallydouble-stranded.
 16. The method of claim 14 or 15, wherein the partiallydouble-stranded nucleic acid domain comprises: (i) a firstoligonucleotide conjugated to the analyte-binding domain; and (ii) ahybridisation oligonucleotide comprising the first hybridisationsequence, the ID sequence and a second hybridisation sequence, the firsthybridisation sequence being located at the 3′ end of the hybridisationoligonucleotide; wherein the double-stranded part of the nucleic aciddomain comprises a duplex between the second hybridisation sequence ofthe hybridisation oligonucleotide and the first oligonucleotide, and thesingle-stranded part of the nucleic acid domain comprises the firsthybridisation sequence of the hybridisation oligonucleotide.
 17. Themethod of claim 16, wherein the hybridisation oligonucleotide comprises,from 5′ to 3′, the second hybridisation sequence, the ID sequence andthe first hybridisation sequence, and the ID sequence is located in thesingle-stranded part of the nucleic acid domain.
 18. The method of claim16 or 17, wherein at least one hybridisation oligonucleotide is extendedin step (iii) to generate the extension product.
 19. The method of anyone of claims 16 to 18, wherein in each proximity probe pair bothnucleic acid domains are partially double-stranded, and one or bothhybridisation oligonucleotides are extended in step (iii) to generatethe extension product.
 20. The method of any one of claims 1 to 19,wherein the nucleic acid domain is a DNA domain.
 21. The method of anyone of claims 1 to 20, wherein in step (iv) the extension product orligation product is amplified by PCR.
 22. The method of any one ofclaims 1 to 21, wherein the extension product or ligation product isdetected by nucleic acid sequencing.
 23. The method of claim 22, whereinprior to sequencing one or more sequencing adapters is attached to theextension product or ligation product in one or more amplificationand/or ligation steps.
 24. The method of claim 23, wherein in step (iv)the extension product or ligation product is amplified in a first PCRreaction wherein a first sequencing adapter is added to one end of theextension product or ligation product; and the product of the first PCRreaction is amplified in a second PCR reaction wherein a secondsequencing adapter is added to the other end of the extension product orligation product.
 25. The method of claim 24, wherein the first PCRreaction is performed with a nucleic acid polymerase that also has 3′ to5′ exonuclease activity, and the second PCR reaction is performed with anucleic acid polymerase that lacks 3′ to 5′ exonuclease activity. 26.The method of any one of claims 24 to 27, wherein prior to sequencing asample index sequence is attached to the extension product or ligationproduct in an amplification or ligation step, preferably wherein thesample index sequence is added to the extension product or ligationproduct during PCR amplification in step (iv).
 27. The method of any oneof claims 22 to 26, wherein the nucleic acid sequencing is massivelyparallel DNA sequencing.
 28. The method of any one of claims 1 to 27,wherein the sample is a plasma or serum sample.
 29. A productcomprising: (i) a plurality of proximity probe pairs, wherein eachproximity probe pair comprises a first proximity probe and a secondproximity probe, and each proximity probe comprises: (a) aprotein-binding domain specific for a protein; and (b) a nucleic aciddomain, wherein both probes within each pair comprise protein-bindingdomains specific for the same protein, and can simultaneously bind tothe protein; and each probe pair is specific for a different protein;wherein the nucleic acid domain of each proximity probe comprises an IDsequence and at least a first hybridisation sequence, wherein the IDsequence of each proximity probe is different; and wherein in eachproximity probe pair, the first proximity probe and the second proximityprobe comprise paired hybridisation sequences; and, optionally (ii) aplurality of splint oligonucleotides, each splint oligonucleotidecomprising hybridisation sequences complementary to each of the pairedhybridisation sequences of a proximity probe pair; wherein thehybridisation sequences of each proximity probe pair are configured suchthat upon binding of the first and second proximity probe to theirprotein, the respective paired hybridisation sequences of the first andsecond proximity probes hybridise to each other or to a splintoligonucleotide; and wherein at least one pair of hybridisationsequences is shared by at least two pairs of proximity probes.
 30. Theproduct of claim 29, wherein the protein-binding domain is an antibodyor fragment thereof.
 31. The product of claim 29 or 30, furthercomprising one or more background probes which do not bind an analyte,said background probes comprising a nucleic acid domain comprising an IDsequence and a hybridisation sequence shared with at least one proximityprobe.
 32. The product of any one of claims 29 to 31, wherein the IDsequences are barcode sequences.
 33. The product of any one of claims 29to 32, wherein at least one pair of hybridisation sequences is unique toa single pair of proximity probes.
 34. The product of any one of claims29 to 33, wherein no more than 10 proximity probe pairs share the samepair of hybridisation sequences.
 35. The product of any one of claims 29to 34, wherein at least 75% of proximity probe pairs share their pair ofhybridisation sequences with another proximity probe pair.
 36. Theproduct of any one of claims 29 to 35, wherein the nucleic acid domainsof each proximity probe pair comprise complementary hybridisationsequences capable of hybridising to one another to form a duplex. 37.The product of any one of claims 29 to 36, wherein the proximity probepairs are as defined in any one of claim 14 to 17 or 20.